Effectiveness of antitoxin therapy in the treatment of botulism and tetanus is significantly reduced, if the treatment is not administered shortly after exposure or onset of disease. Rapid diagnosis and early administration of antitoxin therapy is therefore essential for a patient's recovery. Development of detection devices capable of selectively sensing minute quantities of neurotoxins in body fluids will be critical for providing rapid diagnostic assessment. This proposal outlines a program of research leading to the construction and use of a multidimensional nanofluidic device, specifically designed to manipulate samples, which must be handled in extremely small quantities, e.g., the potent neurotoxins from the Clostridium botulinum and C. tetani families. In the proposed approach, electrokinetic and other standard microfluidic flows are exploited to move reactant and product species among the separate compartments of the device, which are integrated using a new biofluidic device, the molecular gate, based on novel membranes developed at UIUC capable of performing important biomolecular manipulations, e.g. affinity binding and molecular sieving, while simultaneously functioning as the switching elements between microfluidic compartments. The specific aims are: Specific Aim 1. Determine the optimum reaction and separation conditions for the toxins. A set of neurotoxin-specific recognition agents will be developed such that the molecular recognition event results in the generation of highly specific and isolatable reaction products. The separation and detection behavior of these reaction products will be investigated under conditions appropriate to the nanoscale device to be explored in Specific Aims 2 and 3. Specific Aim 2. Incorporate the separation, reaction and detection processes into the micro/nanofluidic device. Miniaturize the molecular recognition, separation, and detection events and incorporate them into a multidimensional hybrid nanofluidic/microfluidic architecture capable of performing sequential chemical manipulations on ultrasmall volumes of complex biomolecular samples. Specific Aim 3. Multiplex the preseparation, molecular recognition, separation, and detection events appropriate to each of the 7 neurotoxins into a single device.